Regensburg, Univ., Diss., 2008

Thesis (Ph. D.)--University of Missouri--Columbia, 1996. / Typescript. Vita. Includes bibliographical references (leaves: 108-124). Also available on the Internet.

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves [110]-121).

Thesis (Ph.D.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 125 pages. Includes Vita. Includes bibliographical references.

Thesis (Ph.D. in Molecular Biology) -- University of Colorado at Denver and Health Sciences Center, 2005. / Typescript. Includes bibliographical references (leaves 126-149). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Universiẗat, Diss., 2003--Düsseldorf.

Orientadores: Marcelo Menossi Teixeira, Agustine Gentile / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-18T02:51:01Z (GMT). No. of bitstreams: 1 Dias_FabioOmetto_M.pdf: 10515001 bytes, checksum: 45aee75247b1cfb2cf161e57b8dcf10c (MD5) Previous issue date: 2011 / Resumo: A transcrição é um evento crucial para a expressão gênica, sendo que a RNA polimerase II tem um papel destacado: são suas subunidades que interagem entre si e com os fatores de transcrição (TFs), formando um complexo que sintetiza o transcrito primário a partir da molécula de DNA. Entender como as subunidades funcionam e interagem entre si e com os TFs é um ponto fundamental para entender as bases da expressão gênica. Uma subunidade intrigante é a quarta maior subunidade, Rpb4, que juntamente com a Rpb7, a sétima maior subunidade, forma um heterodímero localizado em uma região à parte do core enzimático. Em situações de crescimento, Rpb4 encontra-se pouco associada com Rpb7. Porém, em situações de estresse a ligação das subunidades é de suma importância para a viabilidade celular. Esta tese faz a primeira caracterização do gene ScRpb4 de cana-de-açúcar, que codifica uma proteína homóloga à proteína correspondente à quarta maior subunidade da RNA Pol II de plantas. A proteína ScRpb4 foi expressa em Eschericchia coli e os resultados indicam que os maiores níveis foram observados com a construção baseada em pET-28a à 37 °C, induzidas por 4 horas com IPTG. A expressão do gene ScRpb4 foi uniforme em diferentes tecidos e condições, possuindo um ligeiro aumento da expressão na folha imatura, gema lateral, raiz e flor. Na hibridização in situ verificou-se que os transcritos foram encontrados na zona periférica do sistema vascular de folhas e no meristema apical das inflorescências, mostrando que ScRpb4 e expresso em tecidos imaturos com ativa divisão celular. A localização subcelular revelou que ScRpb4 está presente tanto no núcleo quanto no citoplasma, fato similar ao previamente reportado em leveduras. Todos os resultados encontrados estão de acordo com o provável papel de Rpb4 como parte da maquinaria transcricional, e trazem novos dados para essa subunidade em plantas / Abstract: Transcription is a crucial event for gene expression, whereas RNA Polymerase II plays an important role: its subunits interacts with each other and with the transcription factors (TFs) form a complex that synthesizes the primary transcript from the DNA molecule. Understanding how the subunits work and interact among themselves and with the TFs is a key point to understand the basis of gene expression. An interesting subunit is the fourth largest subunit, Rpb4, which together with Rpb7, the seventh largest subunit, forms a heterodimer located away from the enzymatic core. In yeast, in situations of optimal conditions, Rpb4 is often associated with Rpb7, but in non­optimal conditions, the association of the subunits is critical for cell viability. Our work does the first characterization of the ScRpb4 gene from sugarcane, which encodes a homologous protein to the proteins corresponding to the fourth largest subunit of the RNA Polymerase II from plants. The ScRpb4 protein was expressed in Eschericchia coli and results indicate that the highest levels of expression was observed with the construction based on pET-28a at 37 °C, induced with IPfG for 4 hours. ScRpb4 gene expression was uniform in different tissues and conditions, with a slightly higher expression in immature leaf, lateral bud, root and flowers. In situ hybridization showed that the ScRpb4 transcripts were found at the periphery of the vascular system in leaves and in the apical meristem of inflorescence, showing that ScRpb4 is expressed in immature tissues with active cell division. The subcellular localization revealed that ScRpb4 functions in nucleus and cytoplasm, similarly to previous reports found in yeast. All the results found are in agreement with the alleged role of the ScRpb4 as a part of the transcriptional machine and provide new data of this subunit in plants / Mestrado / Genetica Vegetal e Melhoramento / Mestre em Genética e Biologia Molecular

Abstract The maintenance of integrity of the genome is essential for a cell. DNA repair and faithful DNA replication ensure the stability of the genome. DNA polymerases (pols) are the enzymes that synthesise DNA, a process important both in DNA replication and repair. In DNA replication DNA polymerases duplicate the genome during S phase prior to cell division. Pols α, δ, and ε are implicated in chromosomal DNA replication, but their exact function in replication is not yet completely clear. The mechanisms of different repair pathways and proteins involved are not yet completely characterised either. The deeper understanding of DNA repair and replication mechanisms is crucial for our understanding on the function of the cell. The mechanism of repair of DNA double strand breaks (DSBs) by non-homologous end joining (NHEJ) was studied with an in vitro assay. DNA polymerase activity was found to be involved in NHEJ and important in stabilising DNA ends. Antibodies against pol α, but not pol β or ε, decreased NHEJ significantly, which indicates the involvement of pol α in NHEJ. In addition, the removal of proliferating cell nuclear antigen (PCNA) slightly decreased NHEJ activity. The division of labour between pols α, δ, and ε during DNA replication was studied. Results from UV-crosslinking, chromatin association, replication in isolated nuclei, and immunoelectron microscopy (IEM) studies showed that there are temporal differences between the activities and localisations of the pols during S phase. Pol α was active throughout S phase, pol ε was more active at early S phase, whereas the activity of pol δ increased as S phase advanced. These results suggest that pols δ and ε function independently during DNA replication. Pol ε could be crosslinked to nascent RNA, and this labelling was not linked to DNA replication, but rather to transcription. Immunoprecipitation studies indicated that pol ε, but not pols α and δ, associated with RNA polymerase II (RNA pol II). Only the hyperphosphorylated, transcriptionally active RNA pol II was found to associate with pol ε. A large proportion of pol ε and RNA pol II colocalised in cells as determined with immunoelectron microscopy. The interaction between pol ε and RNA pol II suggests that they are involved in a global regulation of transcription and DNA replication.

No description available.

Coding sequences (5,334 nt total) from elongation factor-1α, elongation factor-2, and the largest subunit of RNA polymerase II were determined for 6 species of Tardigrada, 2 of Arthropoda, and 2 of Onychophora. Parsimony and likelihood analyses of nucleotides and amino acids yielded strong support for Tardigrada and all internal nodes (i.e., 100% bootstrap support for Tardigrada, Eutardigrada, Parachela, Hypsibiidae, and Macrobiotidae). Results are in agreement with morphology and an earlier molecular study based on analysis of 18S ribosomal sequences. Divergence times have been estimated from amino acid sequence data using an empirical Bayesian statistical approach, which does not assume a strict molecular clock. Divergence time estimates are pre-Vendian for Tardigrada/Arthropoda, Vendian or earlier for Eutardigrada/Heterotardigrada, Silurian to Ordovician for Parachela/Apochela, Permian to Carboniferous for Hypsibiidae and Macrobiotidae, and Mesozoic for Isohypsibius/Thulinia (both within Hypsibiidae) and Macrobiotus/Richtersius (both within Macrobiotidae).